The long-term goals of this project are to advance our knowledge about the functions of H1 linker histones and to understand the functional significance of the diversity present in this family of chromatin proteins. In most eukaryotic cells H1 linker histones are nearly as abundant as nucleosome core particles. Therefore, H1 histones play a key role in the structure of the chromatin fiber. H1 histones affect gene expression as well as many other processes requiring access to DNA. Much of our knowledge about the functions of H1 has been derived from in vitro experiments. Our approach is to analyze the functions of H1 histones in vivo in mice and in embryonic stem (ES) cells. The mouse (and other mammalian) H1 histones consist of at least 8 nonallelic variants or subtypes that differ considerably in their primary sequences and in their expression during development and tissue differentiation. These subtypes offer an additional level of regulation of chromatin function. Our strategy for studying the functions of linker histones has been to generate and characterize mice and ES cell lines in which one or more H1 genes have been inactivated by gene targeting. We have generated a large collection of mouse strains consisting of both single null mutants (6 of the 8 subtypes have been inactivated) and compound null strains. One of our most informative strains has 3 H1 genes inactivated simultaneously (triple knock-out, TKO). This mutant demonstrated that, unlike in less complex eukaryotes, H1 histones are essential for mammalian development. Some of the mutant strains and TKO ES cells have been analyzed for effects on gene expression. The results showed that, contrary to conclusions derived from in vitro experiments, H1 is not a global repressor of transcription. Importantly, we discovered that H1 is involved in promoting DNA methylation at imprinted gene loci. We now propose to use our unique set of mouse strains and ES cell lines to: (1) Understand the mechanism(s) by which H1 controls DNA methylation. We also propose to identify on a genome-wide scale the sites in the genome at which H1 affects DNA methylation. (2) Assess the role of H1 posttranslational modifications and H1 subtype diversity in the ability of H1 to regulate gene expression and DNA methylation. (3) Develop new mutant mouse strains and ES cell lines, specifically a conditional triple H1 null, and use them to study the role of H1 in postnatal development, cell differentiation and tissue-specific gene expression. Our studies suggest that H1 histones are intimately involved in controlling gene expression, DNA methylation, imprinting and other epigenetic modifications. These processes are required for normal development and abnormalities in them are associated with cancer and other human diseases. We think we have a unique opportunity to help understand some of the numerous important functions of this major component of eukaryotic chromosomes. PUBLIC HEALTH RELEVANCE: The goals of this project are to understand the functions of a major component of mammalian chromosomes called H1 linker histone. H1 is involved in many aspects of chromosome function including repair of DNA damage, recombination and silencing of genes and viruses. Thus, H1 is a key intermediate in processes that contribute to the development of numerous human diseases, including cancer.